Partial purification of biologically active, low molecular weight, human antihemophilic factor free of Von Willebrand factor. I. Partial characterization and evidence for disulfide bond(s) susceptible to limited reduction.
Partially purified (approx. 5000-fold), low molecular weight human antihemophilic factor, free of detectable Von Willebrand factor (ristocetin cofactor activity or Von Willebrand antigen), was prepared from fresh citrated plasma by limited reduction with 1 mM dithiothreitol and chromatography on Sepharose CL-4B, Sephadex G-100, and polyelectrolyte E-5. The ratio of antihemophilic factor activity to Von Willebrand factor activity or antigen was greater than 27 000 : 1. The antihemophilic factor activity could be neutralized with homologous antibody and could be further increased with thrombin. The Mr (approx. 116 000) was determined by calibrated gel permeation chromatography, electrophoresis in 5% polyacrylamide gels with sodium dodecyl sulfate and by electrophoresis in large-pore acrylamide gels without it. Since the low Mr antihemophilic factor could be prepared by treating fresh rather than fresh-frozen plasma with dithiothreitol, it was concluded that partial reduction of the antihemophilic factor with this reagent helped to maintain the antihemophilic factor in a low Mr form. When iodo[l-14C]acetamide was used to alkylate the reduced plasma proteins prior to purification, the molecular weight of the purified antihemophilic factor remained low despite numerous purification steps. By this means, one of four radioactive proteins (Mr 116 000) in the final preparation was bound specifically to homologous antihemophilic factor antibody and attributed to 14C-labeled antihemophilic factor. While the data suggest that antihemophilic factor in fresh plasma contains one or more dithiothreitol-sensitive intramolecular disulfide bonds, the possibility of disulfide linkages with other proteins(s) cannot be excluded.
D055598:Chemical Phenomena; D002621:Chemistry; D004220:Disulfides; D004229:Dithiothreitol; D004591:Electrophoresis, Polyacrylamide Gel; D005169:Factor VIII / Q000276:immunology / Q000302:isolation & purification*; D006801:Humans; D008970:Molecular Weight; D010084:Oxidation-Reduction; D013917:Thrombin; D014841:von Willebrand Factor / Q000302:isolation & purification
D004220:Disulfides; D014841:von Willebrand Factor; D005169:Factor VIII; D013917:Thrombin; D004229:Dithiothreitol